We have reported that plasma contains a factor (circulating shock protein, CSP) that induces cellular depolarization and fluid uptake by cells in vitro. Plasma levels of CSP are elevated following hemorrhage in the rate and the dog and sepsis in the rat. These actions of CSP are contrary to the normal compensatory mechanisms that move fluid out of cells and into the interstitium to effect full restitution of blood volume. CSP is a 200 kD protein complex containing a 70-80 kD peptide, which was sequenced and identified as vitronectin (Vn), and a strongly associating 54kD peptide which appears to be the acute phase protein plasminogen activator inhibitor1 (PAI1). PAI1 induces depolarization with an apparent KM of 1000nM, addition of Vn decreases the KM to 100nM and in the presence of Vn and Fe3+ the KM is 1nM. Plasminogen activators (PA) which specifically bind and hydrolyze PAI1, convert PAI1 into a form (PAI1 act) that has greatly enhanced depolarizing activity. The Specific Aims of this project are as follows. 1). We will study the factors that modify production and binding of PAI1 act, determine its identify and isolate its cell surface receptor. We will determine if the production and binding of Vn PAI1 to plasminogen activator forming Vn PAI1 PA, c) the cleavage of PAI1 by PA to release PAI1 act and d) the binding of PAI1act to its cellular receptor. The final amplitude of depolarization and the concentration of PAI1 act may be affected by phosphorylation subsequent hydrolysis, and by ferric iron. Thus, the experiments in this AIM examine the effects of modification of one or another molecule in the complex on the depolarizing activity of PAI1 act. 2). We will determine if a fixed volume hemorrhage or a hemorrhage that is proportional to the prevailing arterial pressure leads to changes in the plasma levels of PAI1 and changes in endothelial PAI1 mRNA, and if these changes are mediated through TNF, IL1 and glucocorticoid.